1. Intracellular antibiotics and Listeria monocytogenes
Paul M. Tulkens, MD, PhD
Hugues Chanteux, PhD
Stéphane Carryn, PhD
Cellular and Molecular Pharmacology
Catholic University of Louvain, Brussels, Belgium
Presented at the 5th European Congress of Chemotherapy, Rhodos, Greece, October 17th-20th, 2003
5th ECC Rhodes, Greece
19/10/2003

2. Why intracellular / intratissular antibiotics ?
The Cell
antibiotic
bacteria
Black Box...
5th ECC Rhodes, Greece
19/10/2003

3. Subcellular localization ?
cytosol
fluoroquinolones
beta-lactams
ansamycins
macrolides (1/3) ?
endosomes
phagosomes
phago- lysososomes
lysososomes
macrolides (2/3)
aminoglycosides
5th ECC Rhodes, Greece
19/10/2003

4. Mechanisms of localisation and accumulation ...
cytosol
fluoroquinolones
Mechanism unknown
(loose binding to
lipid-containing proteins ? …)
5th ECC Rhodes, Greece
19/10/2003

5. Mechanisms of localisation and accumulation ...
proton trapping
binding to phospholipids
for aminoglycosides: inability to cross membranes
lysososomes
macrolides
aminoglycosides
5th ECC Rhodes, Greece
19/10/2003

6. Subcellular bioavailability of antibiotics ?
High Fair Nil
FQ / Ansamyc. / cytosol. ML lysosom. ML / AG
5th ECC Rhodes, Greece
19/10/2003

7. Subcellular bioavailability of antibiotics ?
Fluoroquinolones
move easily
across
membranes FQ
5th ECC Rhodes, Greece
19/10/2003

8. Subcellular bioavailability of antibiotics ?
aminoglycosides
and lysosomal macrolides
reamain largely if not totally sequestered
in an acidic environment ...
5th ECC Rhodes, Greece
19/10/2003

9. Listeria monocytogenes
Illustration:
the Listeria story
antibiotics:
ampicillin/meropenem
azithromycin
sparfloxacin/moxifloxcin
pivampicillin
Listeria monocytogenes
hly+
5th ECC Rhodes, Greece
19/10/2003

10. Intracellular infection cycle of Listeria monocytogenes hly+
from Portnoy et al.
5th ECC Rhodes, Greece
19/10/2003

11. Following the intracellular fate of Listeria m. by EM
in cytosol
escape from vacuole
phagocytosis
5th ECC Rhodes, Greece
19/10/2003

12. 1st question: is there a simple relation between MIC, accumulation and intracellular activity (5 h model)
MIC
AMPI AZ SP
0.0
0.5
1.0
1.5
Accumulation
AMPI AZ SP 25 50 75
100
Activity *
AMPI AZ SP
0.0
0.5
1.0
1.5
*  log CFU 5h Ce = 10 x MIC
Ouadhriri et al., AAC,1999
5th ECC Rhodes, Greece
19/10/2003

13. Listeria m. and ampicillin
Ampicillin is poorly active against intracellular Listeria m. in spite of its favourable MIC;
lack of accumulation ...
Why do you keep ampicillin ?
extracellular bacteria
get intracellular activity with very large doses ?? (but -lactams are NOT dose-dependent…)
but may be you just have to wait ...
5th ECC Rhodes, Greece
19/10/2003

14. -lactams become bactericidal intracellularly after 24h
control
ampicillin 5 10 15 20 25 4 6 7 8 9
Time (h)
CFU / mg protein -5 -4 -3 -2 -1 1 2 3 4
Changee from original inuculum
INTRA 5h
INTRA 24h
EXTRA 24h
Control Ampicillin Meropenm
(Carryn et al., 2002, JAC 51:
5th ECC Rhodes, Greece
19/10/2003

15. Listeria m. and azithromycin
Azitromycin is also poorly active against
intracellular Listeria m. in spite of its exceptionally large intracellular concentration
most azithromycin is trapped in lysosomes azithromycin is poorly bactericidal
Is there a future for macrolides ?
5th ECC Rhodes, Greece
19/10/2003

16. Listeria m. and fluoroquinolones
In this pharmacological model *, sparfloxacin IS the most active in spite of a unfavourable MIC (1.4 µg/ml) and modest cellular accumulation (12 x)
Fluoroquinolones have a large subcellular bioavailability are highly bactericidal
Why don’t you use fluoroquinolones today ?
too low intrinsic activity ** ...
* all Ce = 10 X the MIC
5th ECC Rhodes, Greece
19/10/2003

17. But look at moxifloxacin (5h model) ...
Carryn et al., AAC, 2002
5th ECC Rhodes, Greece
19/10/2003

18. Comparative intracellular activities
1.5
log) D
1.0
ampicillin
moxifloxacin
0.5
0.0
Change from original inoculum (
-0.5
Moxifloxacin is profoundly bactericidal at clinically achievable concentrations
-1.0
-1.5
-2.0
-2.5 25 50 75
100
% of Cmax
(Carryn et al., 2002, AAC 43: )
5th ECC Rhodes, Greece
19/10/2003

19. However, intracellular moxifloxacin is NOT more active intracellularly than extracellularly ...
broth
cells
Carryn et al., AAC, 2002
5th ECC Rhodes, Greece
19/10/2003

20. A basic prodrug of a -lactam ?
pH 7.4
pH 7.0
pH 5.4
Proton pump
ProD
ProD
ProDH+ H+ D
5th ECC Rhodes, Greece
19/10/2003

21. regenerate ampicillin
Basic compounds that
H2N
Phthalimidomethylampicillin (PIMA)
regenerate ampicillin
accumulate in J774 macrophages
Fan et al, Bioorg. Med. Chem. Let.1997
Pivaloyloxymethylampicillin (PIVA)
Paternotte et al, Biorg. Med. Chem. 2001
5th ECC Rhodes, Greece
19/10/2003

22. Intracellular activity for extracellular concentrations of ...
PIVA
PIMA
10X MIC
0.5X MIC
CTRL
AMPI 1 2 3 4 5 10 6 7 1 2 3 4 5 10 6 7
CFU/mg prot
Time (h)
Same activity for PIVA, PIMA and AMPI
Only PIVA is active
Chanteux et al, JAC, 2003
5th ECC Rhodes, Greece
19/10/2003

23. But maintains it if the medium is renewed every 5 h
At low extracellular concentration, PIVA loses its activity after 5 h if the medium is not renewed 5 10 15 20 4 6 8
CTRL
Extracellular concentration
PIVA
0.5X MIC
CFU/mg prot
But maintains it if the medium is renewed every 5 h
Time (h)
Chanteux et al, JAC, 2003
5th ECC Rhodes, Greece
19/10/2003

24. PIVA releases large amount of intracellular ampicillin
AMPI from PIVA 5 10 15 20 25 30
AMPI from PIVA with changes of medium)
AMPI from PIMA
Cc/Ce
AMPI from AMPI
Time (h)
Chanteux et al, JAC, 2003
5th ECC Rhodes, Greece
19/10/2003

25. The seven pillars of intracellular / intratissular activity ?D* 4
4. Subcell. bioavailability 7
7. Cooper. with host def. D 1
1. Penetration D
3. Accumulation 3 5
5. Expression of activity 2
2. No efflux
6. Bacterial responsiveness
and pharmacodynamics 6
5th ECC Rhodes, Greece
19/10/2003

26. The 6 pillars of intracellular / intratissular accumulation and activity of antibiotics...4 D D 5 2
Françoise Van Bambeke, Stéphane Carryn, Cristina Seral, Hugues Chanteux, Donatienne Tyteca, Marie-Paule Mingeot-Leclercq ...
5th ECC Rhodes, Greece
19/10/2003